The term reactive oxygen species (ROS) is a generalized description for a collection of reactive oxygen molecules of biological significance. These include: superoxide (O2), hydroxyl radical (OH.), peroxyl radical (ROO), alkoxyl radical (RO), hydroperoxyl radical (HOO), hypochlorous acid (HOCl), hydrogen peroxide (H2O2), ozone (O3), singlet oxygen (1O2) and peroxinitrite (ONOO).
ROS are often though of as being directly detrimental to cell viability because they can irreversibly damage key macromolecules such as proteins, nucleic acids and lipids. however there is now evidence that low levels of at least one ROS, hydrogen peroxide (H2O2) has been implicated in the regulations of signal transduction pathways linked to the control of cell proliferation, cell growth and cell death.
ROS, particularly those with mild oxidant capabilities, form suitable signaling molecules as they are capable of oxidizing the reduced thiol groups of cysteine residues to form disulfide bonds with glutathione, an adjacent cysteine residue or a small protein such as thioredoxin. This mild and reversible oxidation is referred to as thiol group modification. As ROS levels increase, more thiol groups become oxidized to disulfides. Consequently, the ratio of reduced groups (thiols) to oxidized groups (disulfide) is a measure of the redox status of proteins, cells or tissues.
Once oxidised, thiol group modifications can be reversed or reduced by specialised enzyme systems, such as thioredoxin or glutaredoxin. This reversible modification of a protein's cysteines between an oxidised and reduced state is analogous to the regulation of a protein's function by phosphorylation/dephosphorylation. Changes in the redox status of a protein, involving disulfide formation and glutathionylation, have been shown to affect the activity of several different signalling transduction proteins and it is thought that changes in the thiol redox status may influence many aspects of cell function, viability and survival.
Mammalian tissues are rich in protein thiols (20-40 mM) and many intracellular proteins have been identified that can undergo thiol group modification. However, despite great interest, only in a few cases has the biological significance of these modifications been identified. This is in part due to a poor understanding of the complexity of the system, a lack of knowledge of the relationship between the thiol redox system and other antioxidant systems and the difficulty in identifying specific biological effects. For example, it is difficult to establish that specific thiol group modifications exist in vivo and are not simply a manifestation of the unnatural oxidising conditions of in vitro systems.
Furthermore, the current methods for determining the redox status of biological systems e.g. proteins, cells, tissues in vivo (and even in vitro) lack sufficient sensitivity, reproducibility or specificity and do not allow the detailed investigation of the effects of changes in redox status on cell function, viability and survival. The current methods fall into two main categories and are discussed further below.
Methods Based on Total Redox Changes
One method involves reacting reduced thiol groups of proteins with groups such as DTNB or Ellman's reagent (for colorimetric determination), bromobimane (for fluorescence based determination) or groups that result in signal amplification (papain). However, most (greater than 90%) of the cysteine residues on a protein are in the reduced thiol form. Therefore, techniques must be very sensitive to detect the difference between, for example, 90% reduced cysteine and 95% reduced cysteine. A more sensitive assessment of thiol redox changes comes from measuring the oxidised disulfides, but this increases the complexity of the method. Furthermore, such methods of measuring oxidised disulfides are not particularly precise and are technically demanding and time consuming.
A second approach to measuring changes in thiol groups is indirect and involves assessing the ratio of oxidised glutathione (GSSG) to reduced glutathione (GSH). Glutathione is the substrate for several antioxidant enzymes. The underlying assumption is that changes in the GSSG/GSH ratio will reflect changes in the reduction status of the cysteine groups on a protein. However, the glutathione system can act independently of the thioredoxin thiol reduction system. Furthermore, the GSSG/GSH analytical techniques are not suitable for the analysis of mitochondria, cells or tissues where only limited sample is available.
Additionally, the above methods rely on the determination of the relative abundance of reduced or oxidised cysteine residues relative to the amount of protein in the sample. The relatively poor precision of protein assays reduces the precision in assessing thiol redox changes.
Methods Involved with Measuring Specific Protein Changes
A third approach to the analysis of thiol redox changes uses a “one label” approach and polyacrylamide gel electrophoresis (PAGE). Generally, this involves attaching or reacting one label, such as a radioactive label, to the reduced thiol groups and measuring the relative amount of labelled residues compared to the total amount of protein. Alternatively, the reduced thiol groups initially present on a protein are blocked and then the protein is exposed to a reducing agent. A label, such as a radioactive label, is then attached to the thiol groups that have been generated following the reduction of any groups that were initially in the oxidised disulfide state. However, this method does not allow the concurrent measurement of both the reduced and oxidised cysteine residues of a protein.
The lack of precision of this approach is further accentuated by variations inherent with PAGE and other systems for visualising the results of the methods. This greatly reduces the practical utility of these methods where only one signal is measured at a time.
The present invention seeks to address or at least partially ameliorate the problems attendant with the prior art.